This invention relates to the preparation of antibodies, particularly monoclonal antibodies secreted by somatic cell hybridomas, to nucleic acids, particularly DNA.multidot.RNA hybrids. Such antibodies are useful in the isolation, detection, and quantitation of nucleic acid duplexes. In particular, the antibodies can be used in nucleic acid hybridization assays to detect hybrids formed between a polynucleotide sequence of interest in a test sample and a known complementary probe.
The detection of specific polynucleotide sequences by the analytical hybridization technique is useful in the fields of recombinant DNA, human and veterinary medicine, agriculture, and food science, among others. In particular, the technique can be used to detect and identify etiological agents such as bacteria and viruses, to screen bacteria for antibiotic resistance, to aid in the diagnosis of genetic disorders such as sickle cell anemia an thalassemia, and to detect cancerous cells.
The state-of-the-art nucleic acid hybridization assay techniques generally involve immobilization of the sample nucleic acid on a solid support followed by hybridization with a labeled form of a complementary probe polynucleotide. Hybridization between particular base sequences or genes of interest in the sample nucleic acid and labeled probe is determined by separating the solid support from the remainder of the reaction mixture which contains unbound labeled probe, followed by detection of the label on the solid support. The preparation of labeled probe generally involves chemical modification of the probe nucleic acid to attach or form a detectable chemical group.
The necessity of immobilizing sample nucleic acids and/or chemically modifying the probe in order to perform conventional hybridization methods poses several significant problems. The procedures required to accomplish immobilization are generally time consuming and add a step which is undesirable for routine use of the technique in a clinical laboratory. Proteins and other materials in the heterogeneous sample, particularly in the case of clinical samples, can also interfere with the immobilization of the nucleic acids. Further, the large-scale preparation of labeled probes normally involves complicated and expensive synthetic and purification procedures. Because the labeled polynucleotide must retain its ability to sensitively hybridize with its complementary sequence of interest, the availability of useful synthetic approaches is severely limited. The synthesis of labeled probes by the methods of nick translation, end labeling, second strand synthesis, and reverse transcription involve enzymatic steps which add the further requirement that the modified or labeled nucleotides must serve as effective substrates for the polymerase enzymes that assemble the labeled polynucleotide. Direct chemical modification of the polynucleotide is also possible, however, such methods are generally quite inefficient and can alter the ability of the labeled polynucleotide to hybridize to the complementary sequence.
As alternatives to immobilizing sample nucleic acids and adding labeled probe, one can use an immobilized probe and label the sample nucleic acids in situ, or one can use a dual hybridization technique requiring two probes, one of which is immobilized and the other labeled [Methods in Enzymology 65:468 (1968) and Gene 21:77-86 (1983)]. The former alternative, however, is even less desirable since the in situ labeling of the sample nucleic acids requires a high degree of technical skill which is not routinely found in clinical technicians and there are no simple, reliable methods for monitoring the labeling yield, which can be a significant problem if the labeling media contain variable amounts of inhibitors of the labeling reaction. The dual hybridization technique has the disadvantages of requiring an additional reagent and incubation step and the kinetics of the hybridization reaction can be slow and inefficient. The accuracy of the assay can also be variable if the complementarity of the two probes with the sample sequence is variable.
Techniques for directly detecting the polynucleotide duplex formed as the product of hybridization between the sample and probe polynucleotides, and thereby dispensing with the chemical labeling and immobilization of sample or probe polynucleotides, have been generally unsatisfactory. Attempts to generate antibodies which will selectively bind double stranded DNA hybrids over single stranded DNA have generally failed [Parker and Halloran, "Nucleic Acids in Immunology", ed. Plescia and Braun, Springer-Verlag, (New York 1969) pp. 18 et seq]. However, there is a recent report of a monoclonal antibody specific for double stranded native DNA with no indication as to its affinity (European Patent Publication No. 135,159). Hybridization formats are described which in principle eliminate the need for chemical modification of probe DNA for labeling purposes. These methods will suffer from high background signal due to immobilization of nonspecific ds-DNA which is ubiquitous in most test samples and in hybridization solutions.
Some success has been achieved in generating polyclonal antibodies that will bind DNA.multidot.RNA mixed hybrids or RNA.multidot.RNA hybrids and have low affinity for the single stranded polynucleotides [see, for example, Rudkin and Stollar, Nature 265:472 (1977); Van Prooijen-Knegt et al., Exp. Cell Res. 141:397(1982); Reddy and Sofer, Biochem. Biophys. Res. Commun. 103:959 (1981); and Nakazato, Biochem. 19:1835 (1980)].
The polyclonal antiserums contain antibodies to single stranded polynucleotides and double stranded RNA as well as antibodies to DNA.multidot.RNA duplexes. Pure antibody specific for DNA.multidot.RNA would have to be isolated from antiserum or fractionated antiserum by immunoadsorption which is practical only on a small scale. Preparations obtained by immunoadsorption would usually contain immunoglobulins which would not bind DNA.multidot.RNA and these reduce the quality of labeled antibody preparations. These preparations also would contain a mixture of antibodies with various affinities. The quality of antibodies obtained would vary between bleedings from one animal and among antiserums from different animals.
Monoclonal antibody technology can provide a means to select an antibody with desired affinity and specificity. Since hybridomas can be preserved and propagated for long periods, the reproducibility of antibody quality is assured. Stuart et al., PNAS (USA) 78:3751 (1981) immunized mice with a poly(A).multidot.poly(dT) duplex and isolated a hybridoma that produced an antibody to DNA.multidot.RNA. No data is provided concerning the specificity of this monoclonal antibody or its affinity for various DNA.multidot.RNA duplexes. Since it was prepared against a DNA.multidot.RNA homopolymer [poly(A).multidot.poly(dT)], it would be expected to have significantly less specificity and affinity for DNA.multidot.RNA heteropolymer duplexes.